ABSTRACT ATP is one of the most versatile biological molecules in all organisms. It provides readily available energy for almost all enzymatic reactions and phosphorylates proteins and enzymes in almost all signal transduction cascades. It also functions as an extracellular messenger. Although all of these biological processes cannot function without ATP, ATP has almost been taken for granted in performing these essential functions, and research focuses have been much more on other molecules involved in these reactions. Recent research has shown that ATP plays some very important but previously unrecognized functions in cancer. Intratumoral extracellular ATP (eATP) is found to be more than 1,000 times higher than that in normal tissues, in the range of 100-500 M, but the sources, destination and functions of the high levels of eATP are still largely unclear. Our recent studies have revealed that eATP can function either extracellularly or intracellularly because eATP can be internalized by cancer cells by different types of endocytotic processes, particularly macropinocytosis. Once inside cells, ATP elevates intracellular ATP levels; promotes cell metabolism, growth and proliferation; enhances signal transduction for cell survival and proliferation; and augments drug resistance. However, exactly how eATP is internalized and then released by cancer cells is far from fully understood. It is also presently unknown if the eATP internalization is a general phenomenon and phenotype among different cancer types. Furthermore, how and if internalized eATP-induced drug resistance is regulated at transcriptional and translational levels is largely unclear. There is a big gap in our current understanding of eATP and our ultimate goal of more effectively treating cancer by disrupting eATP traffic and functions. The long-term research goal of my lab is to understand how eATP affects cancer at molecular, cellular and animal levels and use gained knowledge to design novel therapies to more effectively treat cancer. The objectives of this proposed study are to determine the prevalence and mechanisms that control and regulate ATP's cross-membrane internalization and intracellular release, and drug resistance in cancer. The central hypothesis of this study is that eATP is internalized by most cancer types and then released inside cells to perform diverse functions such as promoting cell survival and drug resistance not only at the signaling level but also at transcriptional and translational levels. To test this hypothesis, three specific aims are proposed: Specific Aim 1. Determine the prevalence of eATP internalization among difference cancer cell lines and cancer types. Specific Aim 2. Use various ATP analogs and endosome markers to study mechanisms of internalization and release of eATP in cancer cells. Specific Aim 3. Use reverse genetics and bioluminescence imaging to study eATP-mediated drug resistance in cancer cells and xenografted tumors in mice. The completion of the study will provide insights into how eATP gets into cancer cells and performs functions such as promotion of drug resistance, and identification of novel targets for reducing drug resistance and improving cancer patients' survival.